Dual action, inhaled formulations providing both an immediate and sustained release profile

ABSTRACT

Methods for formulating immediate and sustained release anti-infectives and delivery of such for treatment of respiratory tract infections and other medical conditions, and devices and formulations used in connection with such are described.

CROSS-REFERENCE

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 12/261,946, filed Oct. 30, 2008 (currentlypending), which is a continuation-in-part of application of U.S. patentapplication Ser. No. 11/876,539 filed Oct. 22, 2007 (currently pending),which claims the benefit of priority to U.S. Provisional PatentApplication Ser. No. 60/862,753 filed Oct. 24, 2006, all of which areincorporated herein by reference in their entirety noting that thecurrent application controls to the extent there is any contradictionwith any earlier application and to which applications we claim priorityunder 35 USC §120.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions forinhalation such as for treating respiratory tract infections caused by avariety of microorganisms. In particular, the present invention relatesto a bi-phasic release formulation which provides for immediate andsustained release of a drug such as anti-infectives delivered byinhalation for the treatment of cystic fibrosis.

BACKGROUND OF THE INVENTION

Respiratory tract infections are caused by a variety of microorganisms.Infections which are persistent have a myriad of consequences for thehealth care community including increased treatment burden and cost, andfor the patient in terms of more invasive treatment paradigms andpotential for serious illness or even death. It would be beneficial ifan improved treatment paradigm were available to provide prophylactictreatment to prevent susceptible patients from acquiring respiratorytract infections as well as increasing the rate or effectiveness oferadicating the infections in patients already infected with themicroorganisms.

In particular, cystic fibrosis (CF) is one example of a disease in whichpatients often acquire persistent or tenacious respiratory tractinfections. CF is a life-threatening genetic disease affectingapproximately 30,000 people in the United States with a frequency ofapproximately one in every 2,500 live births (Fitzsimmons S C, 1993).The name cystic fibrosis refers to the characteristic scarring(fibrosis) and cyst formation within the pancreas, first recognized inthe 1930s. About 1,000 new cases of CF are diagnosed each year. Morethan 80 percent of patients are diagnosed by age three; however, nearly10 percent of newly diagnosed cases are age 18 or older.

The primary CF defect is expressed as altered ion transport via thecystic fibrosis transmembrane conductance regulator (CFTR), which is theprotein regulating cyclic-AMP-mediated chloride conductance at theapical membranes of secretory epithelia (Schroeder S A et al., 1995).Specifically, the normal release of intracellular chloride intoextracellular fluids fails to respond to normal cAMP elevation. Thisimpaired release of chloride results in the dehydration of surroundingrespiratory and intestinal mucosal linings and impaired sodiumreabsorption of the sudoriferous glands. This mucosal dehydration,coupled with inflammatory and infective by-products, creates a thick andtenacious mucus that clogs and damages airways. Prompt, aggressivetreatment of CF symptoms can extend the lives of those with the disease.

Although most people without CF have two working copies of the CFTRgene, only one is needed to prevent cystic fibrosis. CF develops whenneither gene works normally. Therefore, CF is considered an autosomalrecessive disease. There are more than 1,500 different genetic mutationsassociated with the disease (CFTR mutation database, 2006), thus makinghomozygous and heterozygous screening procedures difficult (Zielenski Jet al., 1995). However, approximately two thirds of the mutations arefound to be delta F508, making it the most common CF mutation (CFGenetic Analysis Consortium, 1994).

The ongoing treatment of CF depends upon the stage of the disease andthe organs involved. Clearing mucus from the lungs is an important partof the daily CF treatment regimen. Chest physical therapy is one form ofairway clearance, and it requires vigorous percussion (by using cuppedhands) on the back and chest to dislodge the thick mucus from the lungs.Other forms of airway clearance can be done with the help of mechanicaldevices used to stimulate mucus clearance. Other types of treatmentsinclude: Pulmozyme®, an inhaled mucolytic agent shown to reduce thenumber of lung infections and improve lung function (Hodson M, 1995);TOBI® (tobramycin solution for inhalation), an aerosolizedaminoglycoside antibiotic used to treat lung infections and also shownto improve lung function (Weber A et al., 1994); and oral azithromycin,a macrolide antibiotic shown to reduce the number of respiratoryexacerbations and the rate of decline of lung function (Wolter J et al.,2002).

As discussed above, high rates of colonization and the challenge ofmanaging Pseudomonas aeruginosa infections in patients with cysticfibrosis (CF) have necessitated a search for safe and effectiveantibiotics. Currently, therapy with an aminoglycoside in combinationwith a beta-lactam or a quinolone antibiotic is the standard. A 96-weekseries of clinical studies including 520 patients withmoderate-to-severe CF showed that patients receiving inhaled tobramycinspent 25 to 33% fewer days in the hospital and experienced significantincreases in lung function (Moss R B, 2001). These results demonstratethe effectiveness of inhaled antibiotics to treat CF. However, thedevelopment of drug resistant strains, especially P. aeruginosa, is amajor concern with the long-term delivery of aerosolized antibiotics viainhalation (LiPuma JJ, 2001).

While azithromycin possesses activity against Staphylococcus aureus,Haemophilus influenzae, and Streptococcus pneumoniae, it has no directactivity against Pseudomonas aeruginosa, Burkholderia cepacia, or othergram-negative non-fermenters (Lode H et al., 1996). Tobramycin possessesactivity against P. aeruginosa; however, the increase in the number ofpatients with resistant isolates on continuous therapy from ˜10% to 80%after 3 months (Smith A L et al., 1989) has led to the intermittentdosing regimen of 28-days-on followed by 28-days-off therapy. Even onintermittent inhaled tobramycin therapy, the percentage of patients withmultiresistant P. aeruginosa increased from 14% at baseline to 23% after18 months of treatment (LiPuma JJ, 2001). The development of atherapeutic regimen that delivers the anti-infective therapy in acontinuous fashion, while still inhibiting the emergence of resistantisolates, may provide an improved treatment paradigm. It is noteworthythat chronic P. aeruginosa airway infections remain the primary cause ofmorbidity and mortality in CF patients. When patients experiencepulmonary exacerbations, the use of antipseudomonal therapy, frequentlyconsisting of a β-lactam and an aminoglycoside, may result in clinicalimprovement and a decrease in bacterial burden. Eradication of theinfection, however, is quite rare.

In CF airways, P. aeruginosa initially has a non-mucoid phenotype, butultimately produces mucoid exopolysaccharide and organizes into abiofilm, which indicates the airway infection has progressed from acuteto chronic. Bacteria in biofilms are very slow growing due to ananaerobic environment and are inherently resistant to antimicrobialagents, since sessile cells are much less susceptible than cells growingplanktonically. It has been reported that biofilm cells are at least 500times more resistant to antibacterial agents (Costerton J W et al.,1995). Thus, the transition to the mucoid phenotype and production of abiofilm contribute to the persistence of P. aeruginosa in CF patientswith chronic infection by protecting the bacteria from host defenses andinterfering with the delivery of antibiotics to the bacterial cell.

Although much effort has been made to improve the care and treatment ofindividuals with CF, and the average lifespan has increased, the medianage of survival for people with CF is only to the late 30s (CFFoundation web site, 2006). Thus, a continuing need exists for improvedformulations of anti-infectives, especially for administration byinhalation. The present invention addresses this need.

Ciprofloxacin is a fluoroquinolone antibiotic that is indicated for thetreatment of lower respiratory tract infections due to P. aeruginosa,which is common in patients with cystic fibrosis. Ciprofloxacin is broadspectrum and, in addition to P. aeruginosa, is active against severalother types of gram-negative and gram-positive bacteria. It acts byinhibition of topoisomerase II (DNA gyrase) and topoisomerase IV, whichare enzymes required for bacterial replication, transcription, repair,and recombination. This mechanism of action is different from that forpenicillins, cephalosporins, aminoglycosides, macrolides, andtetracyclines, and therefore bacteria resistant to these classes ofdrugs may be susceptible to ciprofloxacin. Thus, CF patients who havedeveloped resistance to the aminoglycoside tobramycin (TOBI), can likelystill be treated with ciprofloxacin. There is no known cross-resistancebetween ciprofloxacin and other classes of antimicrobials.

Despite its attractive antimicrobial properties, ciprofloxacin doesproduce bothersome side effects, such as GI intolerance (vomiting,diarrhea, abdominal discomfort), as well as dizziness, insomnia,irritability and increased levels of anxiety. There is a clear need forimproved treatment regimes that can be used chronically, withoutresulting in these debilitating side effects.

Delivering ciprofloxacin as an inhaled aerosol has the potential toaddress these concerns by compartmentalizing the delivery and action ofthe drug in the respiratory tract, which is the primary site ofinfection.

Currently there is no aerosolized form of ciprofloxacin with regulatoryapproval for human use, capable of targeting antibiotic delivery directto the area of primary infection. In part this is because the poorsolubility and bitterness of the drug have inhibited development of aformulation suitable for inhalation. Furthermore, the tissuedistribution of ciprofloxacin is so rapid that the drug residence timein the lung is too short to provide additional therapeutic benefit overdrug administered by oral or IV routes.

Phospholipid vehicles as drug delivery systems were rediscovered as“liposomes” in 1965 (Bangham et al., 1965). The therapeutic propertiesof many active pharmaceutical ingredients (APIs) can be improved byincorporation into liposomal drug delivery systems. The general termliposome covers a wide variety of structures, but generally all arecomposed of one or more lipid bilayers enclosing an aqueous space inwhich drugs can be encapsulated. The liposomes applied in this programare known in the drug delivery field as large unilamellar vesicles(LUV), which are the preferred liposomal structures for IV drugadministration.

Liposome encapsulation improves biopharmaceutical characteristicsthrough a number of mechanisms including altered drug pharmacokineticsand biodistribution, sustained drug release from the carrier, enhanceddelivery to disease sites, and protection of the active drug speciesfrom degradation. Liposome formulations of the anticancer agentsdoxorubicin (Myocet®/Evacet®, Doxyl®/Caelyx®), daunorubicin (DaunoXome®)the anti-fungal agent amphotericin B (Abelcet®, AmBisome®, Amphotec®)and a benzoporphyrin (Visudyne®) are examples of successful productsintroduced into the US, European and Japanese markets over the lastdecade. Furthermore, a number of second-generation products have been inlate-stage clinical trials, including Inex's vincristine sulphateliposomes injection (VSLI). The proven safety and efficacy oflipid-based carriers make them attractive candidates for the formulationof pharmaceuticals.

Therefore, in comparison to the current ciprofloxacin formulations, aliposomal ciprofloxacin aerosol formulation should offer severalbenefits: 1) higher drug concentrations, 2) increased drug residencetime via sustained release at the site of infection, 3) decreased sideeffects, 4) increased palatability, 5) better penetration into thebacteria, and 6) better penetration into the cells infected by bacteria.It has previously been shown that inhalation of liposome-encapsulatedfluoroquinolone antibiotics may be effective in treatment of lunginfections. In a mouse model of F. tularensis liposomal encapsulatedfluoroquinolone antibiotics were shown to be superior to the free orunencapsulated fluoroquinolone by increasing survival (CA2,215,716,CA2,174,803, and CA2,101,241).

Another application, EP1083881B1, describes liposomes containing adrug-conjugate comprising a quinolone compound covalently attached to anamino acid. Yet another application, U.S. Publication No. 20004142026,also describes the use of liposome-encapsulated antibiotics and thepotential for administration of a lower dose of a liposome-encapsulatedanti-infective, by a factor of 10 or 100, than for the freeunencapsulated anti-infective.

It has also been reported that the presence of sub-inhibitoryconcentrations of antibiotic agents within the depths of the biofilmwill provide selective pressures for the development of more resistantphenotypes (Gilbert P et al., 1997). This may be partly due to thefailure of the antibiotics to penetrate the glycocalyx adequately.

SUMMARY OF THE INVENTION

An aspect of the invention is an aerosolized, bi-phasic, formulationwhich is aerosolized to create a bi-phasic aerosol of inhaled dropletsor particles. The droplets or particles comprise a free drug (e.g., ananti-infective compound) in which drug is not encapsulated and which maybe ciprofloxacin. The particles further comprise a liposome whichencapsulates a drug such as an anti-infective compound which also may beciprofloxin. The free and liposome encapsulated drug are included withina pharmaceutically acceptable excipient which is formulated foraerosolized delivery. The particles may further include an additionaltherapeutic agent which may be free and/or in a liposome and which canbe any pharmaceutically active drug which is different from the firstdrug.

The formulation and the resulting particles created when the formulationis aerosolized are comprised of a pharmaceutically acceptable carrier,free drug, and drug encapsulated within liposomes. In some situationsthe pharmaceutically acceptable carrier can be completely eliminatedsuch as when the free drug is in a liquid state. However, the carrier isgenerally necessary to provide a solvent for the free drug and thatsolvent may be water, ethanol, a combination of water and ethanol orother useful solvents. The percentage of solvent in the formulation mayvary from 0% to 90% but is generally kept at a level which issufficiently high to maintain the drug in solution at the pH of theformulation. That level will vary from drug to drug and vary as the pHvaries. The carrier can be present in the formulation in an amount of10%, 20%, 30%, 40%, 50%, 60% etc. or more or any incremental amountsthere in between.

The formulation includes the drug in two different forms. First, thedrug is in a free form which is either liquid or dissolved in a solvent.Second, the drug is encapsulated in liposomes. The ratio of the freedrug to the drug encapsulated in liposomes can vary. Generally, the freedrug makes up 5%, 10%, 20%, 30%, etc. up to 80% of the formulation. Thedrug present within the liposome makes up the remaining percentage ofdrug present in the formulation. Thus, drug present in the liposomes canbe present in the amount of from 20% up to 95% of the total drug presentin the formulation.

The formulation may have a pH of 7.4±20%. In some aspects of theinvention the formulation is prepared at a relatively low pH such as 5.4and allowed to adjust to a pH of about 7.4 after it is delivered.Alternatively, the formulation can be formulated at a high pH of about9.4 and allowed to adjust downward to 7.4 after administration.

The formulation includes liposomes which have the encapsulatedpharmaceutically active drug therein which liposomes are designed toprovide for controlled release of the drug. Controlled release of thisaspect in the invention indicates that the drug may be released in anamount of about 0.1% to 100% per hour over a period of time of 1-24hours or 0.5% to 20% per hour over a period of time of 1-12 hours, oralternatively, releases about 2% to 10% per hour over a period of timeof about 1 to 6 hours. Incremental amounts in terms of the percentage ofthe drug and the number of hours which are between the ranges providedabove in half percentage amounts and half hour amounts and otherincremental amounts are intended to be encompassed by the presentinvention. One aspect of the invention is a formulation comprisingliposomes which are delivered via an aerosol to the lungs of a humanpatient, the liposomes comprising free and encapsulated ciprofloxacin orother anti-infective agent. The liposomes may be unilamellar ormultilamellar, and may be bioadhesive, containing a molecule such ashyaluronic acid. At least one therapeutic agent in addition to the freeand liposome-encapsulated anti-infective may also be included in thecomposition. That therapeutic agent may be free drug or encapsulateddrug present with a pharmaceutically acceptable carrier useful fordirect inhalation into human lungs. The other drugs may include enzymesto reduce the viscoelasticity of the mucus such as DNase or othermucolytic agents, chemicals to upregulate the chloride ion channel orincrease flow of ions across the cells, including lantibiotics such asduramycin, agents to promote hydration or mucociliary clearanceincluding epithelial sodium channel (ENaC) inhibitors or P2Y2 agonistssuch as denufosol, elastase inhibitors including Alpha-1 antitrypsin(AAT), bronchodilators, steroids, N-acetylcysteine, interferon gamma,interferon alpha, agents that enhance the activity of the antibioticagainst biofilm bacteria such as sodium salicylate (Polonio R E et al.,2001), or antibiotics known to those skilled in the art. Inflammationand constriction of the airways are also associated with cystic fibrosisand its treatment. Accordingly, bronchodilators, such as β₂-adrenergicreceptor agonists and antimuscarinics, and anti-inflammatory agents,including inhaled corticosteroids, non-steroidal anti-inflammatories,leukotriene receptor antagonists or synthesis inhibitors, and others,may also be combined with an anti-infective.

A further aspect of the invention is a method for treating cysticfibrosis in a patient, the method comprising administering a formulationcomprising the anti-infective; e.g., ciprofloxacin, encapsulated inliposomes to the patient. The formulation is preferably administered byinhalation to the patient.

According to another aspect of the present invention, a formulationcomprising both a free and encapsulated anti-infective provides aninitially high therapeutic level of the anti-infective in the lungs toovercome the barrier to eradicate the difficult to treat biofilmbacteria, while maintaining a sustained release of anti-infective overtime. While some aspects of biofilm resistance are poorly understood,the dominant mechanisms are thought to be related to: (i) modifiednutrient environments and suppression of growth rate within the biofilm;(ii) direct interactions between the exopolymer matrices, and theirconstituents, and antimicrobials, affecting diffusion and availability;and (iii) the development of biofilm/attachment-specific phenotypes(Gilbert P et al., 1997). The intent of the immediate-releaseanti-infective; e.g., ciprofloxacin, is thus to rapidly increase theantibiotic concentration in the lung to therapeutic levels around thedifficult to eradicate biofilm bacteria to address the challenges oflower diffusion rate of antibiotic to and within the biofilm. Thesustained-release anti-infective; e.g., ciprofloxacin, serves tomaintain a therapeutic level of antibiotic in the lung thereby providingcontinued therapy over a longer time frame, increasing efficacy,reducing the frequency of administration, and reducing the potential forresistant colonies to form. The high levels of free antibiotic may alsoserve to attenuate the cytotoxic response to the presence of liposomes(i.e., particles) in the lung and thus reduce the influx of activatedmacrophages.

According to another aspect of the present invention, the immediaterelease of high levels of an anti-infective may allow enhancedpenetration of the glycocalyx. The sustained release of theanti-infective may ensure that the anti-infective agent never fallsbelow the sub-inhibitory concentration and so reduces the likelihood offorming resistance to the anti-infective.

Although ciprofloxacin is a particularly useful anti-infective in thisinvention, there is no desire to limit this invention to ciprofloxacin.Other antibiotics or anti-infectives can be used such as those selectedfrom the group consisting of: an aminoglycoside, a tetracycline, asulfonamide, p-aminobenzoic acid, a diaminopyrimidine, a quinolone, a.beta.-lactam, a .beta.-lactam and a .beta.-lactamase inhibitor,chloraphenicol, a macrolide, penicillins, cephalosporins,corticosteroid, prostaglandin, linomycin, clindamycin, spectinomycin,polymyxin B, colistin, vancomycin, bacitracin, isoniazid, rifampin,ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin,a sulfone, clofazimine, thalidomide, a polyene antifungal, flucytosine,imidazole, triazole, griseofulvin, terconazole, butoconazole ciclopirax,ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine, orany combination thereof.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the formulations and methodology as more fully describedbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects and embodiments of the invention are best understood from thefollowing detailed description when read in conjunction with theaccompanying drawings. It is emphasized that, according to commonpractice, the various features of the drawings are not to-scale. On thecontrary, the dimensions of the various features are arbitrarilyexpanded or reduced for clarity. Included in the drawings are thefollowing figures:

FIG. 1 is a manufacturing flow chart of liposomal ciprofloxacin forinhalation (HSPC/Chol—10 L Batch).

FIG. 2 is a graph showing the cumulative survival rate of mice followinginfection with P. aeruginosa-laden agarose beads on Day 0. Mice weretreated intranasally once daily starting on Day 2 and ending on Day 9with the liposomal formulation of ciprofloxacin (drug) at one of threedifferent concentrations (100%, open diamond; 33%, closed square; or10%, open triangle). Diluent was used as a control (closed circle).Surviving mice were sacrificed on Day 10.

FIG. 3 is a bar graph showing increases in levels of IL-1 following 14days of inhalation of either control (Saline), low dose liposomalciprofloxacin (Low), high dose liposomal ciprofloxacin (High),combination of free and liposomal ciprofloxacin (Mixture) and freeciprofloxacin (Free). The dark bars on the graph show the levels allreturn to baseline values after the 28 day recovery evaluation.

DETAILED DESCRIPTION OF THE INVENTION

Before the present method of formulating ciprofloxacin-encapsulatedliposomes and delivery of such for prevention and/or treatment of cysticfibrosis and other medical conditions, and devices and formulations usedin connection with such are described, it is to be understood that thisinvention is not limited to the particular methodology, devices andformulations described, as such methods, devices and formulations may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aformulation” includes a plurality of such formulations and reference to“the method” includes reference to one or more methods and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

As used herein, anti-infective refers to agents that act againstinfections, such as bacterial, viral, fungal, mycobacterial, orprotozoal infections.

Anti-infectives covered by the invention include but are not limited toquinolones (such as nalidixic acid, cinoxacin, ciprofloxacin andnorfloxacin and the like), sulfonamides (e.g., sulfanilamide,sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and thelike), aminoglycosides (e.g., streptomycin, gentamicin, tobramycin,amikacin, netilmicin, kanamycin, and the like), tetracyclines (such aschlortetracycline, oxytetracycline, methacycline, doxycycline,minocycline and the like), para-aminobenzoic acid, diaminopyrimidines(such as trimethoprim, often used in conjunction with sulfamethoxazole,pyrazinamide, and the like), penicillins (such as penicillin G,penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin,carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin,piperacillin, and the like), penicillinase resistant penicillin (such asmethicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin and thelike), first generation cephalosporins (such as cefadroxil, cephalexin,cephradine, cephalothin, cephapirin, cefazolin, and the like), secondgeneration cephalosporins (such as cefaclor, cefamandole, cefonicid,cefoxitin, cefotetan, cefuroxime, cefuroxime axetil, cefinetazole,cefprozil, loracarbef, ceforanide, and the like), third generationcephalosporins (such as cefepime, cefoperazone, cefotaxime, ceftizoxime,ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, and thelike), other beta-lactams (such as imipenem, meropenem, aztreonam,clavulanic acid, sulbactam, tazobactam, and the like), beta-lactamaseinhibitors (such as clavulanic acid), chloramphenicol, macrolides (suchas erythromycin, azithromycin, clarithromycin, and the like),lincomycin, clindamycin, spectinomycin, polymyxin B, polymixins (such aspolymyxin A, B, C, D, E.sub.1 (colistin A), or E.sub.2, colistin B or C,and the like) colistin, vancomycin, bacitracin, isoniazid, rifampin,ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin,sulfones (such as dapsone, sulfoxone sodium, and the like), clofazimine,thalidomide, or any other antibacterial agent that can be lipidencapsulated. Anti-infectives can include antifungal agents, includingpolyene antifungals (such as amphotericin B, nystatin, natamycin, andthe like), flucytosine, imidazoles (such as miconazole, clotrimazole,econazole, ketoconazole, and the like), triazoles (such as itraconazole,fluconazole, and the like), griseofulvin, terconazole, butoconazoleciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,terbinafine, or any other antifungal that can be lipid encapsulated orcomplexed and pharmaceutically acceptable salts thereof and combinationsthereof. Discussion and the examples are directed primarily towardciprofloxacin but the scope of the application is not intended to belimited to this anti-infective. Combinations of drugs can be used.

Bronchodilators covered by the invention include but are not limited toβ₂-adrenergic receptor agonists (such as albuterol, bambuterol,salbutamol, salmeterol, formoterol, arformoterol, levosalbutamol,procaterol, indacaterol, carmoterol, milveterol, procaterol,terbutaline, and the like), and antimuscarinics (such as trospium,ipratropium, glycopyrronium, aclidinium, and the like). Combinations ofdrugs may be used.

Anti-inflammatories covered by the invention include but are not limitedto inhaled corticosteroids (such as beclometasone, budesonide,ciclesonide, fluticasone, etiprednol, mometasone, and the like),leukotriene receptor antagonists and leukotriene synthesis inhibitors(such as montelukast, zileuton, ibudilast, zafirlukast, pranlukast,amelubant, tipelukast, and the like), cyclooxygenase inhibitors (such asibuprofen, ketoprofen, ketorolac, indometacin, naproxen, zaltoprofen,lomoxicam, meloxicam, celecoxib, lumiracoxib, etoricoxib, piroxicam,ampiroxicam, cinnoxicam, diclofenac, felbinac, lornoxicam, mesalazine,triflusal, tinoridine, iguratimod, pamicogrel, and the like).Combinations of drugs may be used.

As used herein, “Formulation” refers to the liposome-encapsulatedanti-infective, with any excipients or additional active ingredients,either as a dry powder or suspended or dissolved in a liquid.

The terms “subject,” “individual,” “patient,” and “host” are usedinterchangeably herein and refer to any vertebrate, particularly anymammal and most particularly including human subjects, farm animals, andmammalian pets. The subject may be, but is not necessarily under thecare of a health care professional such as a doctor.

A “stable” formulation is one in which the protein or enzyme thereinessentially retains its physical and chemical stability and integrityupon storage and exposure to relatively high temperatures. Variousanalytical techniques for measuring peptide stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991), andJones, A. (1993) Adv. Drug Delivery Rev. 10:29-90. Stability can bemeasured at a selected temperature for a selected time period.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theclaimed methods and compositions.

INVENTION IN GENERAL

Ciprofloxacin is a well-established and extensively utilizedbroad-spectrum fluoroquinolone antibiotic that is indicated for thetreatment of lower respiratory tract infections due to P. aeruginosa,which is common in patients with cystic fibrosis. The primary advantageof inhaled antimicrobials is that they target antibiotic delivery to thearea of primary infection and bypass GI-related side effects; however,the poor solubility and bitterness of the drug have limited developmentof a formulation suitable for inhalation. Furthermore, the rapid tissuedistribution of ciprofloxacin means a short drug residence time in thelung thus limiting therapeutic benefit over oral or IV drugadministration. A liposome-encapsulated formulation of ciprofloxacindecreases the limitations and improves management of pulmonaryinfections due to P. aeruginosa pulmonary infections in patients with CFthrough improved biopharmaceutical characteristics and mechanisms suchas altered drug PK and biodistribution, sustained drug release from thecarrier, enhanced delivery to disease sites, and protection of theactive drug species from degradation.

Preclinical studies demonstrate the efficacy of liposomal ciprofloxacinin a biofilm inhibitory concentration assay using clinical isolates ofPseudomonas aeruginosa, a CF mouse model of P. aeruginosa lung infectionand in a mouse model of Francisella tularensis lung infection. Safetypharmacology studies of respiration in rats and dogs of inhaledliposomal ciprofloxacin found no marked effects upon respiratoryparameters. These studies showed that the inhalation of levels of “free”ciprofloxacin together with the liposomal ciprofloxacin formulationallowed the dosing of greater amounts of liposomal ciprofloxacin withoutobservation of focal macrophage accumulation or increases in theproinflammatory cytokine IL-1β in the lung fluid. This finding is moregenerally applicable to other liposomal formulations delivered to thelung. These results are also applicable to pharmaceutical preparationsor formulations which otherwise may result in increases in macrophageaccumulation in the lung or the increase in proinflammatory cytokines inthe lung in response to the therapy.

The invention includes a formulation that combines ciprofloxacin (or adifferent immune blunting agent; e.g., zithromax) with another drug;e.g., liposomal ciprofloxacin, delivered via the inhalation route. Theliposomal encapsulated ciprofloxacin may be substituted with anantibiotic other than ciprofloxacin and may be formulated without usingliposomes. The other drug does not have to be an antibiotic and may beany drug that is believed to have some beneficial properties whendelivered to the lung.

The invention is not limited to the treatment of CF patients. In fact,there are many patients and indications for which this therapy may bebeneficial, including bronchiectasis, pneumonia, and other lunginfections. This treatment paradigm would also apply to other lungdiseases including COPD, asthma, pulmonary hypertension and others inwhich a formulation of free ciprofloxacin is delivered in combinationwith another drug to allow higher dosing of the other drug or saferadministration of the other drug.

The invention also relates to the use of inhaled free ciprofloxacin (ora different immune blunting agent; e.g., zithromax) in combination withother drugs given via inhalation. These other drugs may include anucleotide sequence which may be incorporated into a suitable deliveryvector such as a plasmid or viral vector. The other drug may be atherapeutic nucleotide sequence (DNA, RNA, siRNA), enzymes to reduce theviscoelasticity of the mucus such as DNase and other mucolytic agents,chemicals to upregulate the chloride ion channel or increase flow ofions across the cells, nicotine, P2Y2 agonists, elastase inhibitorsincluding Alpha-1 antitrypsin (AAT), N-acetylcysteine, antibiotics andcationic peptides, such as lantibiotics, and specifically duramycin,short-acting bronchodilators (e.g., β2-adrenergic receptor agonists likealbuterol or indacaterol), M3 muscarinic antagonists (e.g., ipatropiumbromide), K+-channel openers, long-acting bronchodilators (e.g.,formoterol, salmeterol), steroids (e.g., budesonide, fluticasone,triamcinolone, beclomethasone, ciclesonide, etc.), xanthines,leukotriene antagonists (e.g., montelukast sodium), phosphodiesterase 4inhibitors, adenosine receptor antagonists, other miscellaneousanti-inflammatories (e.g., Syk kinase inhibitors (AVE-0950), tryptaseinhibitors (AVE-8923 & AVE-5638), tachykinin antagonists (AVE-5883),inducible nitric oxide synthase inhibitors (GW-274150) and others),transcription factor decoys, TLR-9 agonists, antisense oligonucleotides,siRNA, DNA, CGRP, lidocaine, inverse β₂-agonists, anti-infectiveoxidative therapies, cytokine modulators (e.g., CCR3 receptorantagonists (GSK-766994, DPC-168, AZD-3778), TNF-α production inhibitors(LMP-160 & YS-TH2), and IL-4 antagonists (AVE-0309)), small moleculeinhibitors of IgE, cell adhesion molecule (CAM) inhibitors, smallmolecules targeting the VLA4 receptor or integrin α4β1 (e.g., R-411,PS-460644, DW-908e, & CDP-323), immunomodulators including those thatblock T-cell signaling by inhibition of calcineurin (Tacrolimus),heparin neutralizers (Talactoferrin alfa), cytosolic PLA2 inhibitors(Efipladib), or combinations thereof. The delivery of the combinationproducts may be achieved by combining the drugs into one stableformulation, or providing the drugs in separate containers to becombined at the time of administration or alternatively by sequentiallydelivering the products.

I. Generation of Liposomes Containing Ciprofloxacin

According to aspects of the instant invention, a method is provided forformulating ciprofloxacin and other anti-infectives by encapsulatingthese drugs in liposomes. Composed of naturally-occurring materialswhich are biocompatible and biodegradable, liposomes are used toencapsulate biologically active materials for a variety of purposes.Having a variety of layers, sizes, surface charges and compositions,numerous procedures for liposomal preparation and for drug encapsulationwithin them have been developed, some of which have been scaled up toindustrial levels. Liposomes can be designed to act as sustained releasedrug depots and, in certain applications, aid drug access across cellmembranes.

The sustained release property of the liposomes may be regulated by thenature of the lipid membrane and by the inclusion of other excipients inthe composition of the liposomes. Extensive research in liposometechnology over many years has yielded a reasonable prediction of therate of drug release based on the composition of the liposomeformulation. The rate of drug release is primarily dependent on thenature of the phospholipids, e.g. hydrogenated (--H) or unhydrogenated(--G), or the phospholipid/cholesterol ratio (the higher this ratio, thefaster the rate of release), the hydrophilic/lipophilic properties ofthe active ingredients and by the method of liposome manufacturing.

Methods for making bioadhesive liposomes can be found, for example, inU.S. Pat. No. 5,401,511 which is incorporated herein by reference in itsentirety along with the patents and publications cited therein whichdescribe liposomes and methods of making liposomes. In recent years,successful attempts have been made to bind different substances toliposomes. For example, binding of chymotrypsin to liposomes has beenstudied as a model for binding substances to liposomal surfaces.Recognizing substances, including antibodies, glycoproteins and lectins,have been bound to liposomal surfaces in an attempt to confer targetspecificity to the liposomes.

The number and surface density of the discrete sites on the liposomalsurfaces for covalent bonding are dictated by the liposome formulationand the liposome type. The liposomal surfaces also have sites fornoncovalent association. Covalent binding is preferred as noncovalentbinding might result in dissociation of the recognizing substances fromthe liposomes at the site of administration since the liposomes and thebioadhesive counterparts of the target site (that is, the bioadhesivematter) compete for the recognizing substances. Such dissociation wouldreverse the administered modified liposomes into regular, non-modifiedliposomes, thereby defeating the purpose of administration of themodified liposomes.

To form covalent conjugates of recognizing substances and liposomes,crosslinking reagents have been studied for effectiveness andbiocompatibility. Once such reagent is glutaraldehyde (GAD). Through thecomplex chemistry of crosslinking by GAD, linkage of the amine residuesof the recognizing substances and liposomes is established.

The crosslinking reagents can include glutaraldehyde (GAD) and a watersoluble carbodiimide, preferably,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The recognizingsubstances include gelatin, collagen, and hyaluronic acid (HA).Following these methodologies, recognizing substances may be utilized asan adhesive or glue to attach the liposomes onto a target area, such asthe lung.

Thus, while not essential to the instant invention, the use of suchbioadhesive liposomes, particularly those having hyaluronic acid as thebioadhesive ligand, will potentially increase residence time inpulmonary sites, and reduce mucociliary clearance and macrophage uptake.

In general, ciprofloxacin is preferably used in the formulations of theinstant invention, although other antibiotics or anti-infectives knownto those skilled in the art may be used.

Multilamellar vesicles (MLV) are prepared according to techniques wellknown in the art. Briefly, in an embodiment, lipids are weighed anddissolved in a suitable organic solvent (such as chloroform orchloroform-methanol mixtures). The organic solvent is evaporated tocomplete dryness in a rotary evaporator, under low pressure, and at atemperature range of about 37-40° C. Following evaporation, theciprofloxacin solution is added to the dry lipid film. The system isvigorously mixed, then incubated for about two hours in, for example, ashaker bath at a temperature range appropriate for the lipidcomposition. The preparation is then preferably buffered, for example,by adding about a one tenth volume of ten-fold concentrated phosphatebuffered saline (PBS), of pH 7.4.

In an embodiment, MLV generated as described above serve as the sourcematerial for acidic unilamellar vesicles (ULV). For example, MLV areprepared as described above and subjected to extrusion in a device suchas, for example, that manufactured by Lipex Biomembranes, Inc.(Vancouver, British Columbia). Extrusion is performed through a seriesof membranes with progressively-smaller pore sizes, such as, forexample, starting with pore sizes in the range of 0.8 to 1.0 μm (one totwo extrusion cycles per pore size) and ending at the pore size rangeselected according to the desired liposome size (e.g., about sevencycles of extrusion at the final pore size).

Exemplary liposome compositions and methods of making them are disclosedin U.S. Pat. Nos. 6,890,555; 6,855,296; 6,770,291; 6,759,057; 6,623,671;6,534,018; 6,355,267; 6,316,024; 6,221,385 and 6,197,333 all of whichare incorporated herein by reference. The liposomes of the invention maybe multilamellar, unilamellar, or any configuration known such asdescribed in the above patents. The liposomes of the instant inventionare preferably made from biocompatible lipids. In general, the size ofthe liposomes generated is over a wide range depending on mode ofdelivery, e.g. 1 nm to 10 μm or 20 nm to 1 μm or about 100 nm indiameter ±20% for pulmonary delivery.

II. Pharmaceutical Formulation of Ciprofloxacin-Containing Liposomes

In a preferred embodiment, the liposome-encapsulated ciprofloxacin isadministered to a patient in an aerosol inhalation device but could beadministered by the IV route. In some embodiments, ciprofloxacin isencapsulated in the liposomes in combination with other pharmaceuticalsthat are also encapsulated. In some embodiments, ciprofloxacin isencapsulated in the liposomes in combination with other pharmaceuticalsthat are not encapsulated. In some embodiments, the liposomes areadministered in combination with ciprofloxacin that is not encapsulated,with pharmaceuticals that are not encapsulated, or various combinationsthereof.

Formulations of the invention can include liposomes containingciprofloxacin in combination with an amount of alveolar surfactantprotein effective to enhance the transport of the liposomes across thepulmonary surface and into the circulatory system of the patient. U.S.Pat. No. 5,006,343, issued Apr. 9, 1991, which is incorporated herein byreference, disclosed liposomes and formulations of liposomes used inintrapulmonary delivery. The formulations and methodology disclosed inU.S. Pat. No. 5,006,343 can be adapted for the application ofciprofloxacin and may be included within the delivery device of thepresent invention in order to provide for effective treatments of cysticfibrosis patients.

Regardless of the form of the drug formulation, it is preferable tocreate droplets or particles for inhalation in the range of about 0.5 μmto 12 μm, preferably 1 μm to 6 μm, and more preferably about 2-4 μm. Bycreating inhaled particles which have a relatively narrow range of size,it is possible to further increase the efficiency of the drug deliverysystem and improve the repeatability of the dosing. Thus, it ispreferable that the particles not only have a size in the range of 0.5μm to 12 μm or 2 μm to 6 μm or about 3-4 μm but that the mean particlesize be within a narrow range so that 80% or more of the particles beingdelivered to a patient have a particle diameter which is within ±20% ofthe average particle size, preferably ±10% and more preferably ±5% ofthe average particle size.

The formulations of the invention may be administered to a patient usinga disposable package and portable, hand-held, battery-powered device,such as the AERx device (U.S. Pat. No. 5,823,178, Aradigm, Hayward,Calif.). Alternatively, the formulations of the instant invention may becarried out using a mechanical (non-electronic) device. Other inhalationdevices may be used to deliver the formulations including conventionaljet nebulizers, ultrasonic nebulizers, soft mist inhalers, dry powderinhalers (DPIs), metered dose inhalers (MDIs), condensation aerosolgenerators, and other systems. The proportion of free ciprofloxacin toencapsulated ciprofloxacin was shown to remain constant afternebulization; i.e., there was no damage to the liposomes duringnebulization that would result in premature release of a portion of theencapsulated antibiotic. This finding is unexpected based upon priorliterature reports (Niven R W and Schreier H, 1990) but ensures that theanimal or human inhaling the aerosol will get a reproducible proportionof free to encapsulated drug depositing throughout the lung.

An aerosol may be created by forcing drug through pores of a membranewhich pores have a size in the range of about 0.25 to 6 microns (U.S.Pat. No. 5,823,178). When the pores have this size the particles whichescape through the pores to create the aerosol will have a diameter inthe range of 0.5 to 12 microns. Drug particles may be released with anair flow intended to keep the particles within this size range. Thecreation of small particles may be facilitated by the use of thevibration device which provides a vibration frequency in the range ofabout 800 to about 4000 kilohertz. Those skilled in the art willrecognize that some adjustments can be made in the parameters such asthe size of the pores from which drug is released, vibration frequency,pressure, and other parameters based on the density and viscosity of theformulation keeping in mind that an object of some embodiments is toprovide aerosolized particles having a diameter in the range of about0.5 to 12 microns.

The liposome formulation may be a low viscosity liquid formulation. Theviscosity of the drug by itself or in combination with a carrier shouldbe sufficiently low so that the formulation can be forced out ofopenings to form an aerosol, e.g., using 20 to 200 psi to form anaerosol preferably having a particle size in the range of about 0.5 to12 microns.

In an embodiment, a low boiling point, highly volatile propellant iscombined with the liposomes of the invention and a pharmaceuticallyacceptable excipient. The liposomes may be provided as a suspension ordry powder in the propellant, or, in another embodiment, the liposomesare dissolved in solution within the propellant. Both of theseformulations may be readily included within a container which has avalve as its only opening. Since the propellant is highly volatile, i.e.has a low boiling point, the contents of the container will be underpressure.

In accordance with another formulation, the ciprofloxacin-containingliposomes are provided as a dry powder by itself, and in accordance withstill another formulation, the ciprofloxacin-containing liposomes areprovided in a solution formulation. The dry powder may be directlyinhaled by allowing inhalation only at the same measured inspiratoryflow rate and inspiratory volume for each delivery. The powder may bedissolved in an aqueous solvent to create a solution which is movedthrough a porous membrane to create an aerosol for inhalation. Anyformulation which makes it possible to produce aerosolized forms ofciprofloxacin-containing liposomes which can be inhaled and delivered toa patient via the intrapulmonary route may be used in connection withthe present invention. Specific information regarding formulations whichcan be used in connection with aerosolized delivery devices aredescribed within Remington's Pharmaceutical Sciences, A. R. Gennaroeditor (latest edition) Mack Publishing Company. Regarding insulinformulations, it is also useful to note the findings of Sciarra et al.,(1976). When low boiling point propellants are used, the propellants areheld within a pressurized canister of the device and maintained in aliquid state. When the valve is actuated, the propellant is released andforces the active ingredient from the canister along with thepropellant. The propellant will “flash” upon exposure to the surroundingatmosphere, i.e., the propellant immediately evaporates. The flashingoccurs so rapidly that it is essentially pure active ingredient which isactually delivered to the lungs of the patient.

III. Dosing Regimens

Based on the above, it will be understood by those skilled in the artthat a plurality of different treatments and means of administration canbe used to treat a single patient. Thus, patients already receiving suchmedications, for example, as intravenous ciprofloxacin or antibiotics,etc., may benefit from inhalation of the formulations of the presentinvention. Some patients may receive only ciprofloxacin-containingliposome formulations by inhalation. Such patients may have symptoms ofcystic fibrosis, be diagnosed as having lung infections, or havesymptoms of a medical condition, which symptoms may benefit fromadministration to the patient of an antibiotic such as ciprofloxacin.The formulations of the invention may also be used diagnostically. In anembodiment, for example, a patient may receive a dose of a formulationof the invention as part of a procedure to diagnose lung infections,wherein one of more of the patient's symptoms improves in response tothe formulation.

A patient will typically receive a dose of about 0.01 to 10 mg/kg/day ofciprofloxacin ±20% or ±10%. This dose will typically be administered byat least one, preferably several “puffs” from the aerosol device. Thetotal dose per day is preferably administered at least once per day, butmay be divided into two or more doses per day. Some patients may benefitfrom a period of “loading” the patient with ciprofloxacin with a higherdose or more frequent administration over a period of days or weeks,followed by a reduced or maintenance dose. As cystic fibrosis istypically a chronic condition, patients are expected to receive suchtherapy over a prolonged period of time.

It has previously been shown that inhalation of liposome-encapsulatedfluoroquinolone antibiotics may be effective in treatment of lunginfections and were shown to be superior to the free or unencapsulatedfluoroquinolone in a mouse model of F. tularensis (CA 2,215,716, CA2,174,803 and CA 2,101,241). However, they did not anticipate thepotential benefit of combining the free and encapsulated fluoroquinoloneantibiotics to treat those lung infections. According to one aspect ofthe present invention, high concentrations of an antibiotic aredelivered immediately while also providing a sustained release of thetherapeutic over hours or a day.

Another application, EP1083881B1, describes liposomes containing adrug-conjugate comprising a quinolone compound covalently attached to anamino acid. That application does not foresee the requirement to haveboth an immediate release and sustained release component to treat thoselung infections.

Another application, US 2000142026, also describes the use ofliposome-encapsulated antibiotics. That application discusses thepotential for administration of a lower dose of a liposome-encapsulatedantibiotic, by a factor of 10 or 100, than for the free unencapsulatedantibiotic. However, they did not anticipate the benefit of combiningboth free and encapsulated antibiotic to provide an initially hightherapeutic level of the antibiotic in the lungs to overcome the barrierto eradicating the difficult to treat biofilm bacteria.

Other applications describing the use of liposomes containinganti-infectives to treat cystic fibrosis, US 20060073198 and US20040142026 show efficacy for a lower dose liposomal formulationcompared to the use of the free drug alone.

Thus, as discussed above, the formulations according to some aspects ofthe invention include free or non-encapsulated ciprofloxacin incombination with the liposome-encapsulated ciprofloxacin. Suchformulations may provide an immediate benefit with the freeciprofloxacin resulting in a rapid increase in the antibioticconcentration in the lung fluid surrounding the bacterial colonies orbiofilm and reducing their viability, followed by a sustained benefitfrom the encapsulated ciprofloxacin which continues to kill the bacteriaor decrease its ability to reproduce, or reducing the possibility ofantibiotic resistant colonies arising. The skilled practitioner willunderstand that the relative advantages of the formulations of theinvention in treating medical conditions on a patient-by-patient basis.

It has long been known that inhalation of fine particulate matter (PM)from environmental and occupational sources can be associated with anincrease in respiratory symptoms including cough, wheeze, shortness ofbreath (Koren 1995), reduction in lung function (Pope et al., 1995),exacerbation of asthma (Choudhury et al., 1997) and COPD (Abbey et al,1995), and even increased premature mortality. Nurkiewicz et al (2004)have shown that pulmonary exposure to fine PM impairs endotheliumdependent dilation in systemic arterioles and more recently thatultrafine PM was more toxic by virtue of its increased surface area(2008). In those studies in rats, the principal histopathologicalterations in the lung consisted of particle accumulation withinalveolar macrophages, the presence of anuclear macrophages, and anintimate association between particle-laden alveolar macrophages and thealveolar wall. In addition to the particle accumulation, macrophagescontaining particles were frequently intimately associated with thealveolar wall a location, suggestive of, but not diagnostic of,macrophage activation. These results are consistent with long standingdata that shows that particle delivery to the lung results in anincrease in macrophages in rats (Yevich, 1965). The alveolar macrophagesare phagocytic and provide an important defense mechanism againstinhaled pathogens and clearing inhaled dust from the alveoli. However,if the level of exposure is too high, the macrophages can becomesufficiently stimulated resulting in the secretion of mediators thatcause inflammatory changes in the bronchoalveolar region.

The implication of these studies is that any particulate containingformulation, e.g., liposomes containing an anti-infective, may result inan increased particle load in the lung, stimulation of macrophages andthe potential to cause inflammation and lung damage. Thus, it isimportant that the “particulate” formulation is safe and does notproduce an excessive inflammatory response in the lung. As we describein some aspects of the invention, the presence of free ciprofloxacin, orany free antibiotic with similar anti-inflammatory properties, in theformulation, in addition to providing an immediate high level ofantibiotic to treat the lung infection, may also provide a mechanism toattenuate the lung's response to the inhaled liposome or particulatecontaining formulation; i.e., reduce macrophage influx and othercytokines.

IV. Other Formulations or Carriers

Although liposomes have been primarily described as the vehicle toprovide encapsulation of the therapeutic and thus the sustained releaseeffect, there is no intention to limit the formulation to liposomalformulations. A combination of immediate and sustained releaseformulations or carriers of an anti-infective in the lung may beachieved via a multitude of ways including microspheres, polymers, gels,emulsions, particulates or suspensions, either singly or in combination.Some formulations or carriers may have properties that result in closerassociation with the biofilm matrix and these may prove moreadvantageous with respect to increasing the therapeutic levels of theanti-infective proximal to the biofilm bacteria.

Viscous Controlled Release Formulations

An example of a sustained release polymer formulation is the use ofpoly(ortho esters) as the vehicle. For example, see U.S. Pat. Nos.4,304,767, 4,957,998, 5,968,543 and WO 02/092661 as well as Adv. PolymerSci., 107, 41-92 (1993) and references therein. Viscosities of thesecontrolled release polymers were reported to be in the 1,500 cP range(see Biomaterials, 23, 2002, 4397-4404). Considerably higher forces wererequired for higher molecular weight polymers (see Adv. Drug DelReviews, 53, 2001, 45-73).

Novel Liposomes

Various long-circulating liposomes have been prepared by incorporatingglycolipids or other amphiphilic molecules into the lipid bilayer ofconventional liposomes. Vasopressin entrapped in PEGylatedlong-circulating liposomes even remained bioactive one month afterintravenous injection (Woodle et al., 1992).

A new approach, rather than using unilamellar or multilamellarliposomes, is based on the DEPOFOAM system. These multivesicularliposomes (1-100 μm) contain multiple non-concentric internal aqueouscompartments and lead to an increase in the encapsulation efficiency.After subcutaneous injection, the release of encapsulated peptide andprotein was shown to be prolonged up to 7 days for Depolnsulin and up to3 weeks for the DepoLeuprolide® formulation (Ye, Q et al., 2000).

The company Novosom A G has patented a novel liposome-based depot systemfor proteins and peptides. The Cagicles® depots are produced by a twostep method: first, proteins are dissolved in an aqueous medium and thenadded to solutions of membrane-forming substances, which are selectedsuch that the resulting membrane enters into a reversible mutualreaction with the protein. This mild-condition process enables toincrease the encapsulation rate over 30% of incorporated protein.Furthermore, a one month sustained protein release was feasible aftersubcutaneous or intramuscular injection of the Cagicles® depots(Panzner, S., Novosom A G, Application No. 2000-EP11079, WO 2001034115(2000)). These studies have proven the basic applicability of liposomes.The solubility benefits of liposomes are well known and reported.

Lipid Nanoparticles and Microspheres

Solid lipid nanoparticles (SLNs) represent a colloidal carrier systemmainly based on triglycerides. Due to their hydrophobic nature and theirsmall size, SLNs may be more appropriate for incorporation of lipophilicdrugs, which can be easily dissolved in the melted mixture. Forinstance, only small quantities of lysozyme can be incorporated intovarious lipids (Almeida et al., 1997). Solid lipid nanoparticles ownpotential for the encapsulation of drugs with a low solubility (e.g.paclitaxel), for the application of surface-modified SLNs in drugtargeting, or maybe for the use as adjuvant for vaccines. Furthermore,it can be hypothesised that SLNs can be applied for oral drug deliveryin the form of aqueous dispersions or that they can alternatively beused as additives in traditional dosage forms such as tablets, capsulesor pellets.

U.S. Pat. No. 6,277,413 describes a biodegradable microsphere having amatrix, the matrix comprising at least one type of biodegradablepolymer, and at least one type of lipid; and a physiologically activesubstance which is releasable from the biodegradable microsphere.

Lipid Crystals

EP 0767,656B1 describes a pharmaceutical composition, which isglycerol-ester based and contains diacyl glycerol as well asphospholipid(s), or a polar group containing water, glycerol, ethyleneglycol or propylene glycol. The proportions between the components areadjusted to form an L2 phase or a liquid crystalline phase, with thebiological material being dispersed or dissolved in the L2 or liquidcrystalline phase.

Oil Suspensions

Generally, the viscosity of oily media is considerably higher than theviscosity of an aqueous phase such as buffer. Therefore, drug releasecan be prolonged by implementing oil suspensions. In addition, theviscosity of the oily carrier may be further increased by the additionof gelling agents such as aluminum monostearate thus enabling thecontrol of process parameters like drug solubility and drug transferrate. A further important aspect using oils as drug carrier refers tothe distribution coefficient of compounds in the oily medium and thesurrounding tissue. A lipophilic drug with a high distributioncoefficient will primarily accumulate in the oily medium resulting infurther deceleration of effective drug actions.

For several years, various peptides and proteins have been dispersed inoils to engineer sustained-release formulations. Nestor et al. patentedas early as 1979 the development of long-acting injectable depotformulations for super-agonist analogues of luteinizinghormone-releasing hormone (LH-RH), applying oils such as peanut oil orsesame oil and a gelling agent such as aluminum stearate (Nestor et al.,Syntex Inc., U.S. Pat. No. 4,256,737 (1979)).

Hydrogels

Thermoreversible hydrogels are of great interest in drug delivery. Theseinclude thermosensitive gel materials including poly(ethyleneglycol)/poly(propylene glycol) block copolymers (poloxamers),poly(ethylene glycol)/poly(butylenes glycol) block copolymers,poloxamer-g-poly(acrylic acid) and copolymers of Nisopropylacrylamidethat exhibit a sol-to-gel transition in aqueous solutions. Diblockcopolymers of poly(ethylene oxide) (PEG) and poly(lactic acid) (PLA),and triblock copolymers of PEG-PLGA-PEG are also used as alternativehydrogels that would provide biodegradable and injectable drug-deliverysystems under physiological conditions. Some natural polymers includinggelatin, agarose, amylase, amylopectin, cellulose derivatives,carrageenans, and gellan, exhibit thermoreversible gelation behavior.Some cellulose derivatives of natural polymers, such as methyl celluloseand hydroxypropyl cellulose, exhibit reverse thermogelation behavior(gelation at elevated temperatures). Viscosity of these hydrogels is aconcern for parenteral delivery. Viscosity of these hydrogels can beextremely high at low shear rates (Thorgeirsdottir T O et al., 2005).Poly hydroxyl methacralate is extensively used in hydrogel formulations(Peppas et al., 2000). U.S. Pat. No. 6,602,952 describes a polymericstructure comprising a multifunctional poly(alkylene oxide), such as apoly(ethylene glycol) derivative, covalently cross-linked to a polymerselected from the group consisting of chitosan and conjugates ofchitosan and a monofunctional poly(alkylene oxide), such as methoxypoly(ethylene glycol). In aqueous media, the polymeric structure forms ahydrogel.

Depot Formulations

Implantable drug delivery devices provide an attractive therapeutic toolfor treatment of a variety of diseases and conditions, especially when asustained release effect is also added to the therapy. Variousimplantable drug delivery devices have been developed, and are basedupon different mechanisms to accomplish movement of drug from areservoir to the treatment site. U.S. Pat. No. 4,938,763 discloses amethod for forming an implant in situ by dissolving a non-reactive,water insoluble thermoplastic polymer in a biocompatible, water solublesolvent to form a liquid, placing the liquid within the body, andallowing the solvent to dissipate to produce a solid implant. U.S. Pat.No. 5,747,058 describes a composition for the controlled release ofsubstances that includes a non-polymeric non-water solublehigh-viscosity liquid carrier material of viscosity of at least 5,000 cPat body temperature that does not crystallize neat under ambient orphysiological conditions.

Delivery of Macromolecules

The addition of a protein or peptide drug to the anti-infectiveformulation may provide additional therapeutic benefit. A previouslydiscussed example includes Pulmozyme rhDNase which is approved in thetreatment of CF. While some macromolecules may be delivered at a lowdose or at relatively low concentrations, for others it may be necessaryto deliver at high concentrations. Protein formulations at highconcentrations may have physical properties that impact the ability toeasily deliver the protein drug. U.S. Pat. No. 6,541,606 describesprotein crystals or crystal formulations that are encapsulated within amatrix comprising a polymeric carrier to form a composition. Theformulations and compositions enhance preservation of the nativebiologically active tertiary structure of the proteins and create areservoir which can slowly release active protein where and when it isneeded.

Conjugated Systems

Polymer carrier systems may have certain advantages over non-polymericcarriers in terms of avoiding uptake by macrophages. Because liposomesare spherical vesicles made of phospholipids are particles, they gettaken up by macrophages. High levels can be found in the liver andspleen, even when the liposomes are given “stealth” characteristics bycoating them with PEG. Antibodies, meanwhile, have the disadvantage thatmost receptors on tumor cells are also present on normal cells, makingit hard to find ones that are unique to cancer.

In contrast, water-soluble polymers allow working with a single moleculerather than a large particle. To avoid the liver and spleen, unchargedhydrophilic polymers, such as PEG and N-(2-hydroxypropyl)methacrylamidecan be used. When these polymers are hydrated, they can circulate in theblood for periods of up to about 24 hours (C&E News, Volume 80, Number34, 39-47).

Examples of other conjugated systems include PEGylation. PEGylationdecreases the rate of clearance from the bloodstream by increasing theapparent molecular weight of the molecule. Up to a certain size, therate of glomerular filtration of proteins is inversely proportional tothe size of the protein. Decreased clearance can lead to increasedefficiency over the non-PEGylated material (Conforti et al. 1987 andKatre et al. 1987). The conjugation could be either in-vitro or in-vivo.

WO2005034909A2 describes a hyperbranched polymer attached to a core anda biologically active moiety. The biologically active moiety is attachedto the core by means of a substantially non-enzymatically cleavablelinker L. The composition can be used to deliver the biologically activemoiety to its target. U.S. Pat. No. 6,946,134 describes therapeuticproteins fused to albumin or fragments or variants of albumin, thatexhibit extended shelf-life and/or extended or therapeutic activity insolution. The role of albumin as a carrier molecule and its inert natureare desirable properties for use as a carrier and transporter ofpolypeptides in vivo. The use of albumin as a component of an albuminfusion protein as a carrier for various proteins has been suggested inWO93/15199, WO93/15200, and EP413622. The use of N-terminal fragments ofHA for fusions to polypeptides has also been proposed (EP399666).

U.S. Pat. No. 5,367,051 describes fullerene-functionalizedamine-containing polymers and polymerizable monomers characterized byhigh temperature stability, i.e., capable of withstanding a temperatureof at least about 300° C., when in polymerized form. The fullerenegroups are bonded to the polymers through the amine groups on thepolymer.

WO Patent No. 2005073383 describes novel heterodimeric fusion proteinscomprising a first polypeptide including an alpha subunit of FSH (aFSH)linked directly or indirectly to a binding partner of neonatal Fcreceptor (FcRn) and a second polypeptide including a beta subunit of FSH(βFSH) linked directly or indirectly to an FcRn binding partner. Theconjugated polypeptide has increased half-life and bioavailability ascompared to traditional forms of FSH therapy.

Dendrimers

Dendrimers are well-defined polymeric structures. Dendrimers are basedon repeating hyperbranched structures emanating from a central core(U.S. Pat. No. 4,507,466). Typical dendrimers are based onpolyamidoamine (PAMAM), polyethylene imine (PEI), polypropylene imine orpolylysine. These synthetic macromolecules are assembled in a stepwisefashion, with each reaction cycle adding another layer of branches(dubbed “generation”). Dendrimers are synthetically accessed bystepwise, divergent “bottom-up” or convergent “top-down” synthesis.Central structural component is the core unit from which hyperbrancheddendrimers extend in a radially symmetric fashion. The core may provideat least two reactive groups for dendrimer conjugation, it may also beof heterofunctional nature and protecting groups may be used. In thelatter case, the dendrimer may be assembled, and a guest compound may besubsequently conjugated to an anilin core by means of orthogonalchemistries (WO88/01180). The core and dendrimers form the interior orbackbone of a dendrimer. As a consequence of the spherical symmetrysupported by sterical crowding, the terminal groups of the hyperbranchesare defining the exterior. In higher generation dendrimers, the terminalbranches form rather dense shells and flexible internal voids have beendiscovered. It is understood, that for a given dendrimer these cavitiesare filled up by backfolded end groups and tightly coordinated solventmolecules. Dendrimers are related to micelles, similarly well suited tocomplex hydrophobic compounds. But in contrast they exhibit higherstructural order because of their monomolecular nature and the absenceof a dynamic equilibrium of various species. Synthetic compounds canonly diffuse into dendrimers if certain structural requirement such asconformational rigidity and flatness as well as charge distribution suchas affinity to tertiary amines are met. Various apolar compounds such aspyrene or naphthalene have been encapsulated in dendrimers.

In U.S. Pat. No. 5,714,166 and WO95/24221, dendrimer-protein conjugatesare revealed. PAMAM dendrimers of G4 are covalently coupled throughtheir terminal functional groups to insulin, fluorescently labeledinsulin, avidin, monoclonal antibodies and bradykinin. The reactivegroups used for conjugation are only present at the surface of thedendrimers, and therefore any covalent adduct generated by the leachedmethod will be associated with the dendrimer exterior.

PAMAM dendrimers contain free amine groups on their surfaces and readilyassociate with DNA through electrostatic interactions.

WO01/07469 details water-soluble polypeptide dendrimers constituted ofornithin and glycine amino acids. The patent application also teachesthe non-covalent encapsulation of an oligosaccharide, heparin, bydendrimerization of the dendrimer core in presence of heparin under mildconditions. The oligosaccharide is released from the dendrimer bylight-induced cleavage of W-labile bonds within the dendritic backbone.The core structure used here was tris(2-maleimidoethyl) amine. OtherPolymeric Systems

The use of heparin, dextran and methyl methacralate in a biomimetricapproach was evaluated in the development of drug carriers escapingearly capture by phagocytosis (Passirani et al., 1998).

The synthesis of hybrid block and graft copolymers of polyphosphazenesand polystyrene is a way to combine the attributes of both polymers andgenerate new properties. Many of the valuable properties of therespective phosphazene and styrene homopolymers can be combined withoutsacrificing the overall solid state or solution properties of bothpolystyrene and polyphosphazene polymers. U.S. Pat. No. 6,392,008describes such compositions of polyphosphazene-containing polymers.

U.S. Pat. No. 5,176,907 describes biocompatible and biodegradablepoly(phosphoester-urethanes), compositions comprising thepoly(phosphoester-urethanes), and methods of use as a drug deliverydevice and an implant.

V. Combination Therapies

Liposome formulations of the invention may be administered concurrentlywith other drugs as described here. For example, the liposomes of theinvention may be used along with drugs such as DNase, a mucolytic agent,chemicals that up-regulate the chloride ion channel or increase flow ofions across the epithelial surface of cells, a bronchodilator, asteroid, a P2Y2 agonist, an elastase inhibitor such as Alpha-1antitrypsin (AAT), N-acetylcysteine, agents that enhance the activity ofthe antibiotic against biofilm bacteria such as sodium salicylate,interferon gamma, interferon alpha, or a fluoroquinolone selected fromthe group consisting of amifloxacin, cinoxacin, ciprofloxacin,danofloxacin, difloxacin, enoxacin, enrofloxacin, fleroxacin, irloxacin,lomefloxacin, miloxacin, norfloxacin, ofloxacin, pefloxacin, rosoxacin,rufloxacin, sarafloxacin, sparfloxacin, temafloxacin and tosufloxacin oran antibiotic selected from the group of tobramycin, colistin,azithromycin, amikacin, cefaclor (Ceclor), aztreonam, amoxicillin,ceftazidime, cephalexin (Keflex), gentamicin, vancomycin, imipenem,doripenem, piperacillin, minocycline, or erythromycin.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

VI. Method of Treatment

Until now we have discussed primarily the application of this inventionto treat infections in cystic fibrosis patients. However, it will beobvious to one skilled in the art that this invention will have utilityand advantages beyond CF. This method of treatment applies to otherdisease states which involve infections of the nasal passages, airways,inner ear, or lungs; including but not limited to: bronchiectasis,tuberculosis, pneumonia; including but not limited to ventilatorassociated pneumonia, community acquired pneumonia, bronchial pneumonia,lobar pneumonia; infections by Streptococcus pneumoniae, Chlamydia,Mycoplasma pneumonia, staphylococci, prophylactive treatment orprevention for conditions in which infection might arise, e.g.,intubated or ventilated patients, infections in lung transplant patient,bronchitis, pertussis (whooping cough), inner ear infections,streptococal throat infections, inhalation anthrax, tularemia, orsinusitis.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor is it intendedto represent that the experiment below is the only experiment performed.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1 Manufacture of Encapsulated Ciprofloxacin

Ciprofloxacin (50 mg/mL) is encapsulated into liposomes consisting ofhydrogenated soy phosphatidyl-choline (HSPC) (70.6 mg/mL), asemi-synthetic fully hydrogenated derivative of natural soy lecithin(SPC), and cholesterol (29.4 mg/mL). The lipid is organized in abilayer, with an average particle size of 75 to 120 nm. The sterilesuspension is suspended in an isotonic buffer (25 mM histidine, 145 mMNaCl at pH 6.0, 300 mOsm/kg) and administered by inhalation. Theseliposomal ciprofloxacin preparations contained approximately 1%unencapsulated ciprofloxacin.

The manufacturing process includes the following steps.

-   1. Preparation of buffers.-   2. Weighing of lipid components.-   3. Dissolution of lipids in solvent (tBuOH:EtOH:dH2O/49:49:2).-   4. Mixing of the solvent solution of lipids with methylamine    sulphate buffer (10% v/v solvent) to form multilamellar vesicles    (MLVs) with encapsulated methylamine sulphate buffer at 30 mg/mL    lipid.-   5. Extrusion through four stacked 80 nm pore size polycarbonate    filters to generate large unilamellar vesicles (LUVs). A second    extrusion pass was performed to generate liposomes with a mean    diameter of ˜100 nm.-   6. Ultrafiltration to concentrate the liposomes to ˜55 mg/mL total    lipid.-   7. Diafiltration against 10 volumes of buffer (145 mM NaCl, 5 mM    histidine, pH 6.0) to remove ethanol and generate a transmembrane pH    gradient.-   8. Determination of the lipid concentration by HPLC.-   9. Heating of the liposome suspension to 50° C. and slow addition of    powdered ciprofloxacin (60% of the total lipid mass) with stirring.    Ciprofloxacin is added incrementally (10% of mass every 4 minutes    over a 40-minute period) and the product incubated at 50° C. for 20    minutes following addition of the last aliquot to allow completion    of the drug loading process.-   10. Diafiltration of the ciprofloxacin loaded liposomes against    3-volumes of 145 mM NaCl, 5 mM acetate, pH 4.0 to remove    unencapsulated ciprofloxacin under conditions in which the free    ciprofloxacin is soluble.-   11. Diafiltration of the ciprofloxacin loaded liposomes against    5-volumes of 145 mM NaCl, 25 mM histidine, pH 6.0 to remove any    remaining un-encapsulated ciprofloxacin, further reducing the    residual solvent levels and exchanging the external buffer for the    desired final product buffer.-   12. Ultrafiltration of the formulation to a ciprofloxacin    concentration of 50 mg/mL (in-process testing required).-   13. Pre-filtration of the liposomes through 0.45/0.2 μm filter    sheets to remove particulates which can clog sterilizing grade    filters. The filters employed are in fact sterilizing grade filters;    however they are employed at elevated pressures not compatible with    their use for sterile filtration.-   14. Redundant filtration through 0.2 μm sterilizing grade filters.-   15. Sample vialing and packaging.

The overall manufacturing scheme is shown in FIG. 1.

Description of Infection Model:

The ciprofloxacin encapsulated liposomes were evaluated in a mouse modelof P. aeruginosa lung infection. The gut-corrected, Cftr knockout micehave been shown to have a cystic fibrosis lung phenotype followinginfection with P. aeruginosa-laden agarose beads (van Heeckeren et al.,2004), and have a similar inflammatory response as the UNC Cftr knockoutmice (van Heeckeren et al., 2004). All of these features make this thestrain of choice to investigate whether the drug has efficacy in a mousemodel of cystic fibrosis lung infection, and not if there is adifferential response between wild type and cystic fibrosis mice. Miceof one sex, male, were used to eliminate sex as a potential confounder.All mice were between 6-8 weeks of age and weighed >16 g.

P. aeruginosa-laden agarose (PA) beads were made and used, as describedpreviously (van Heeckeren, et al., 1997, van Heeckeren et al., 2000, vanHeeckeren and Schluchter, 2002), with minor differences. Mice wereinoculated with a 1:35 dilution of the beads, and beads were deliveredin mice anesthetized with isoflurane. This was established to be an LD50dose, though subtle differences from experiment to experiment may leadto differential responses, which is not predictable. That is, in oneexperiment the dose is an LD50, but it may be an LD90 in another. Sincewe are interested in investigating whether these drugs have clinicalefficacy, we attempted to dose between the LD50 and the LD90 range ininfected CF control mice. Interventional euthanasia was performed if themice were moribund (severe delay in the righting reflex and palpablycold), and a necropsy performed to determine whether there was overtlung infection. Mice that were sacrificed were included as ifspontaneous death had occurred.

Liposomal Ciprofloxacin Treatments:

Formulations of liposomal ciprofloxacin or sham (diluent) (≦0.05 ml)were delivered intranasally.

Design of Dose-Ranging Study:

Three doses were tested: 10%, 33%, and 100% of full strength (50 mg/ml)ciprofloxacin composed of 99% encapsulated and 1% free ciprofloxacin,plus the liposomal diluent as a negative control. The low- and mid-dosewere prepared by dilution. On Day 0, mice were inoculatedtranstracheally with P. aeruginosa-laden agarose beads diluted 1:35 insterile PBS, pH 7.4. On Days 2 through 9, mice were treated with thedrug or diluent sham once daily. On Day 10, mice were sacrificed. Theoutcome measures included clinical signs (including coat quality,posture, ability to right themselves after being placed in lateralrecumbency, ambulation), changes from initial body weight, and survival.At the time of sacrifice, gross lung pathology was noted,bronchoalveolar lavage (BAL) was performed using 1 ml sterile PBS, pH7.4, whole blood, unprocessed BAL fluid and spleen homogenates weretested for presence or absence of P. aeruginosa, and BAL cells wereenumerated using a hemacytometer.

Survival Results:

FIG. 2 shows the cumulative survival rate for each group out to 10 daysreported as a percentage of the number of mice that survived. At Day 10,the three groups treated with liposomal ciprofloxacin had greatersurvival rates than the diluent control group. There were only 2 deathsin each of the liposomal treatment groups, whereas there were 6 deathsin the diluent group. The 100%-dose group had the longest survival ofall the groups, with all mice surviving out to Day 5, whereas the othergroups all had 2 deaths by this time.

Intranasal administration (to target the lung) of liposome-encapsulatedciprofloxacin containing approximately 1% free ciprofloxacin increasedthe survival rate of mice with P. aeruginosa lung infections.Accordingly, inhaled liposomal ciprofloxacin is efficacious in patientswith cystic fibrosis, or other diseases with lung infections.

FIG. 2 shows the cumulative survival rate following infection. Mice wereinfected with P. aeruginosa-laden agarose beads on Day 0. Mice weretreated intranasally once daily starting on Day 2 and ending on Day 9with the liposomal formulation of ciprofloxacin (drug) at one of threedifferent concentrations (100%, open diamond; 33%, closed square; or10%, open triangle). Diluent was used as a control (closed circle).Surviving mice were sacrificed on Day 10.

Example 2 Preparation of Unencapsulated Ciprofloxacin

A solution of unencapsulated, or “free” ciprofloxacin at a concentrationof 30 mg/mL in 10 mM sodium acetate, pH 3.2, was prepared.

Manufacture of encapsulated ciprofloxacin: Ciprofloxacin (50 mg/mL) wasencapsulated into liposomes consisting of hydrogenated soyphosphatidyl-choline (HSPC) (70.6 mg/mL), a semi-synthetic fullyhydrogenated derivative of natural soy lecithin (SPC), and cholesterol(29.4 mg/mL), as described in Example 1. Characterization of thisliposomal formulation indicated that approximately 1% of theciprofloxacin was free; that is, it was not encapsulated within theliposome.

Description of infection model: Formulations containing freeciprofloxacin and liposome encapsulated ciprofloxacin were evaluated intwo additional experiments in a mouse model of P. aeruginosa lunginfection as described in Example 1.

Design of dose-ranging study: One dose of the combination of free andliposomal ciprofloxacin (0.36 mg/kg free and 0.6 mg/kg liposomalciprofloxacin), two doses of liposomal ciprofloxacin (0.6 mg/kg and 1.2mg/kg) and the liposomal diluent as a negative control were evaluated intwo separate experiments. On Day 0, mice were inoculated transtracheallywith P. aeruginosa-laden agarose beads diluted 1:35 in sterile PBS, pH7.4. On Days 2 through 9, mice were treated with the drug or diluentsham once daily. On Day 10, mice were sacrificed. The outcome measuresincluded clinical signs (including coat quality, posture, ability toright themselves after being placed in lateral recumbency, ambulation),changes from initial body weight, and survival. At the time ofsacrifice, gross lung pathology was noted, bronchoalveolar lavage (BAL)was performed using 1 ml sterile PBS, pH 7.4, whole blood, unprocessedBAL fluid and spleen homogenates were tested for presence or absence ofP. aeruginosa, and BAL cells were enumerated using a hemacytometer.

Survival Results Table 1 shows the cumulative survival rate for eachgroup out to 10 days reported as a percentage of the number of mice thatsurvived from both studies. At Day 10, all groups treated with acombination of free and liposomal ciprofloxacin had greater survivalrates than the diluent control group.

TABLE 1 Mean survival per group from two studies in CF mice with P.aeruginosa lung infection treated with intranasally instilled ARD-3100,or control Mean Mean % Free Starting Mean Survival Dose (mg/kg)Ciprofloxacin Number Mortality (%) 0 (Control) N/A 9 6/9 34% 0.6 1 8.52.5/8.5 66% 1.2 1 8.5   3/8.5 65% 0.96 38 10.5  2.5/10.5 76%Conclusion: Intranasal administration (to target the lung) ofliposome-encapsulated ciprofloxacin increased the survival rate of micewith P. aeruginosa lung infections. Inhaled liposomal ciprofloxacin orcombinations of free and liposomal ciprofloxacin are efficacious inpatients with cystic fibrosis, or other diseases with lung infections.

Example 3 Manufacture of Encapsulated Ciprofloxacin

Ciprofloxacin (50 mg/mL) was encapsulated into liposomes consisting ofhydrogenated soy phosphatidyl-choline (HSPC) (70.6 mg/mL), asemi-synthetic fully hydrogenated derivative of natural soy lecithin(SPC), and cholesterol (29.4 mg/mL), as described in Example 1.Characterization of this liposomal formulation indicated thatapproximately 1% of the ciprofloxacin was free; that is, it was notencapsulated within the liposome.

Delivery of Combination of Free and Encapsulated Ciprofloxacin: Ratherthan using a formulation which contains both free and encapsulatedciprofloxacin, an alternative method is to create the mixture during thedelivery event. For example, the addition of shear or heat in acontrolled fashion may reproducibly result in some of the liposomeslosing their integrity and releasing the contents of drug that werepreviously encapsulated within the liposomes. Studies using theelectromechanical AERx system confirmed the possibility of using thisapproach. Formulations containing approximately 99% encapsulatedciprofloxacin were delivered using the AERx system and the aerosoldroplets were collected. With the temperature controller set attemperatures of 13, 45, 77, 108 and 140° C. the collected aerosolcontained 89, 84, 82, 77 and 41 percent encapsulated, respectively.

The instant invention is shown and described herein in a manner which isconsidered to be the most practical and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

While the instant invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

Example 4 Manufacture of Encapsulated Ciprofloxacin

Ciprofloxacin (50 mg/mL) was encapsulated into liposomes consisting ofhydrogenated soy phosphatidyl-choline (HSPC) (70.6 mg/mL), asemi-synthetic fully hydrogenated derivative of natural soy lecithin(SPC), and cholesterol (29.4 mg/mL), as described in Example 1.Characterization of this liposomal formulation indicated thatapproximately 1% of the ciprofloxacin was free; that is, it was notencapsulated within the liposome.

Manufacture of Unencapsulated (or free) Ciprofloxacin: Under asepticmanufacturing conditions, ciprofloxacin-HCl as powder was dissolved in10 mM sodium acetate solution, pH 3.3 and sterile filtered resulting ina 24 mg/mL solution.

Delivery of Combination of Free and Encapsulated Ciprofloxacin: In thisexample the free and encapsulated formulations are mixed in thenebulizer and delivered as aerosol at the time of administration. The pHof the resulting mixture is between 4.0 and 5.0. Samples of thedelivered aerosol and the solution remaining in the nebulizer followingaerosolization (i.e., the nebulizer residual) were analyzed to ensurethat the mixing and nebulization process did not degrade the integrityof the liposomes. No loss in encapsulated ciprofloxacin was observed.This result ensures that a precise and reproducible amount of free andencapsulated ciprofloxacin is delivered to the human subject or patient,or animals during preclinical safety or preclinical efficacy studies.

Evaluation of the Free and Encapsulated Ciprofloxcain in a 14-DayNose-Only Inhalation Toxicology Study in Sprague Dawley Rats: Variousciprofloxacin formulations were evaluated in this 28 day inhalationtoxicology study with 28 and 56 day recovery arms. Exposure to nebulizedliposomal ciprofloxacin by nose-only inhalation for 6 hours per day, 7days per week for 14 days at a target pulmonary dose of 8.2 (Low), or16.5 (High) mg/kg/day resulted in histopathology findings in the lung(focal macrophage accumulation, and alveolar wall monocyte infiltration)at the end of the 14 day exposure period. After a 28-day recoveryperiod, histopathology findings were still present in the lung (focalmacrophage accumulation, and alveolar wall monocyte infiltration, TableII).

Evaluation of the Free and Encapsulated Ciprofloxcain in a 28-DayNose-Only Inhalation Toxicology Study in Sprague Dawley Rats: Variousciprofloxacin formulations were evaluated in this 28 day inhalationtoxicology study with 28 and 56 day recovery arms. Exposure to nebulizedliposomal ciprofloxacin by nose-only inhalation for 6 hours per day, 7days per week for 28 days at a target pulmonary dose of 11 (Low), 21(Mid), or 31 (High) mg/kg/day resulted in histopathology findings in thelung (diffuse macrophage accumulation, focal macrophage accumulation,and alveolar wall monocyte infiltration) at the end of the 28 dayexposure period. After a 28-day recovery period, histopathology findingswere still present in the lung (focal macrophage accumulation, andalveolar wall monocyte infiltration, Table II). After a 56 day recoveryperiod, histopathology findings were still present in the lung for allthree groups exposed to liposomal ciprofloxacin (focal macrophageaccumulation, and alveolar wall monocyte infiltration, alveolar wallfibrosis and crystal formation in alveolar lumens, Table III).

TABLE II Recovery Necropsy (after 28-day recovery) Group/Mean Lung DoseMixture Mixture 28 Saline Low 14-day Low 28-day High 14-day Mid 28-dayHigh 28-day 14-day day 0 mg/kg 8.2 mg/kg 11 mg/kg 16.5 mg/kg 21 mg/kg 31mg/kg 11.5 mg/kg 34 mg/kg Organ Lesion Sex Incidence (group meanseverity score) Lung Focal M 0/5 (0.00)* 2/5 (0.40) 3/5 (0.60) 5/5(1.40) 5/5 (2.60) 5/5 (2.80) 1/5 (0.20) 3/5 (0.60) macrophageaccumulation Focal F 2/5 (0.40) 4/5 (0.80) 5/5 (2.40) 5/5 (1.20) 5/5(2.60) 5/5 (3.00) 0/5 5/5 (1.60) macrophage accumulation Diffuse M 0/5(0.00) 0/5 0/5 (0.00) 0/5 0/5 (0.00) 0/5 (0.00) 0/5 0/5 (0.00)macrophage accumulation Diffuse F 0/5 (0.00) 0/5 0/5 (0.00) 0/5 0/5(0.00) 0/5 (0.00) 0/5 0/5 (0.00) macrophage accumulation Monocyte M 0/5(0.00) 1/5 (0.20) 0/5 (0.00) 0/5 4/5 (1.00) 5/5 (1.20) 0/5 1/5 (0.20)interstitial infiltration, alveolar wall Monocyte F 0/5 (0.00) 2/5(0.40) 4/5 (1.00) 3/5 (0.60) 5/5 (1.20) 5/5 (1.40) 0/5 4/5 (0.80)interstitial infiltration, alveolar wall Larynx Squamous M 0/5 (0.00)0/5 (0.00) 0/5 (0.00) 0/5 (0.00) 0/5 (0.00) cell hyperplasia- metaplasiaSquamous F 0/4 (0.00) 0/5 (0.00) 0/5 (0.00) 0/5 (0.00) 0/5 (0.00) cellhyperplasia- metaplasia

TABLE III Recovery Necropsy (after 56-day recovery) Group/Mean Lung Dose(Human Dose) Saline Low Mid High Mixture 0 mg/kg 11 mg/kg 21 mg/kg 31mg/kg 34 mg/kg Organ Lesion Sex Incidence (group mean severity score)Lung Focal macrophage M 0/6 (0.00)* 1/5 (0.20) 6/6 (2.33) 6/6 (2.00) 0/6(0.00) accumulation Focal macrophage F 1/6 (0.17) 4/5 (1.40) 6/6 (2.33)6/6 (2.83) 1/6 (0.17) accumulation Diffuse macrophage M 0/6 (0.00) 0/5(0.00) 0/6 (0.00) 0/6 (0.00) 0/6 (0.00) accumulation Diffuse macrophageF 0/6 (0.00) 0/5 (0.00) 0/6 (0.00) 0/6 (0.00) 0/6 (0.00) accumulationMonocyte interstitial M 0/6 (0.00) 0/5 (0.00) 6/6 (1.50) 5/6 (1.17) 0/6(0.00) infiltration, alveolar wall Monocyte interstitial F 0/6 (0.00)2/5 (0.60) 6/6 (1.50) 6/6 (1.67) 0/6 (0.00) infiltration, alveolar wallFibrosis, alveolar wall M 0/6 (0.00) 0/5 (0.00) 5/6 (0.83) 4/6 (0.67)0/6 (0.00) Fibrosis, alveolar wall F 0/6 (0.00) 2/5 (0.40) 5/6 (0.83)5/6 (0.83) 0/6 (0.00) Crystals, alveolar M 0/6 (0.00) 0/5 (0.00) 4/6(1.17) 5/6 (1.17) 0/6 (0.00) lumen Crystals, alveolar F 0/6 (0.00) 2/5(0.40) 5/6 (1.33) 5/6 (1.33) 0/6 (0.00) lumen Larynx Squamous cell M 0/6(0.00) 0/5 (0.00) 0/6 (0.00) 0/6 (0.00) 0/6 (0.00) hyperplasia-metaplasia Squamous cell F 0/6 (0.00) 0/5 (0.00) 0/6 (0.00) 0/6 (0.00)0/6 (0.00) hyperplasia- metaplasia

In contrast, a fourth group of rats exposed to a combination ofencapsulated and free ciprofloxacin for a similar treatment durationexperienced a reduction in histopathology findings (Table II). After the56 day recovery period, the histopathology findings for this group ofrats was not significantly different from controls (Table III).

The explanation for this surprising effect may be that the presence offree ciprofloxacin in the lung attenuates the release of cytokines whichattract macrophages into the lung. The concentration of one cytokinemarker, IL-1, in the lungs of rats after 14 days of inhalation treatmentincreases with increasing doses of liposomal ciprofloxacin (FIG. 3). Theaddition of free ciprofloxacin to the liposomal ciprofloxacinformulation reduces the levels of IL-1 to values similar to those forthe control animals. These findings suggest that the presence of freeciprofloxacin down regulates cytokine markers and reduces the influx ofmacrophages into the lungs.

The instant invention is shown and described herein in a manner which isconsidered to be the most practical and preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

While the instant invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

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1. An aerosolized composition of particles, comprising: a free,unencapsulated first pharmaceutically active drug; a pharmaceuticallyacceptable excipient; and a liposome-encapsulated secondpharmaceutically active drug; wherein the composition is formulated foraerosolized delivery.
 2. The aerosolized composition of claim 1, whereinthe first drug is an anti-infective and the second drug is not ananti-infective.
 3. The aerosolized composition of claim 2, wherein thefirst drug is ciprofloxacin and the second drug is a therapeuticnucleotide sequence.
 4. The aerosolized composition of claim 1, whereinthe first and the second pharmaceutically active drug are each ananti-infective, the particles of the aerosol have a diameter in a rangefrom about 2 microns to about 4 microns and the free drug comprises 5%or more of a total of both free drug and liposome-encapsulated drug inthe formulation.
 5. A pharmaceutical formulation, comprising: apharmaceutically acceptable carrier for aerosolized delivery to humanlungs; a free, unencapsulated anti-infection drug; aliposome-encapsulated anti-infective drug; and a therapeutic nucleotidesequence.
 6. The formulation of claim 5, wherein the free anti-infectivedrug, and the liposome encapsulated anti-infective drug isciprofloxacin.
 7. The formulation of claim 4, wherein the free andliposomal encapsulated anti-infective drug is selected from the groupconsisting of a quinolone, a sulfonamide, an aminoglycoside, atetracycline, para-aminobenzoic acid, a diaminopyrimidine, abeta-lactam, a beta-lactam and a beta-lactamase inhibitor,chloramphenicol, a macrolide, lincomycin, clindamycin, spectinomycin,polymyxin B, colistin, vancomycin, bacitracin, isoniazid, rifampin,ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin,a sulfone, clofazimine, thalidomide, polyene antifungal, flucytosine,imidazole, triazole, griseofulvin, terconazole, butoconazole ciclopirax,ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine andcombinations thereof.
 8. The formulation of claim 5, wherein theliposomes are comprised of a lipid selected from the group consisting offatty acids; lysolipids; sphingolipids; phosphatidylcholines;phosphatidylethanolamines; sphingomyelin; glycolipids; glucolipids;glycosphingolipids; phosphatidic acid; palmitic acid; stearic acid;arachidonic acid; oleic acid; lipids bearing sulfonated mono-, di-,oligo- or polysaccharides; lipids with ether and ester-linked fattyacids, polymerized lipids, diacetyl phosphate, stearylamine,cardiolipin, phospholipids with short chain fatty acids of 6 to 8carbons in length, synthetic phospholipids with asymmetric acyl chains;and lipids bearing a covalently bound polymer.
 9. The formulation ofclaim 5, further comprising: an additional pharmaceutically active drugchosen from a bronchodilator and an anti-inflammatory drug.
 10. Theformulation of claim 9, wherein the additional pharmaceutically activedrug is a bronchodilator chosen from β-adrenergic receptor agonists,antimuscarinics and combinations thereof.
 11. The formulation of claim9, wherein the additional pharmaceutically active drug is anantiinflammatory selected from the group consisting of a corticosteroid,a leukotriene receptor antagonist, a leukotriene synthesis inhibitor,and a cyclooxygenase inhibitor.
 12. The formulation of claim 5, whereinthe liposome-encapsulated anti-infective agent is prepared using aphospholipid selected from the groups consisting ofphosphatidylcholines, lysophosphatidylcholines,phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols,phosphatidic acid, phosphatidylserines, and mixtures thereof; whereinsaid phospholipid is provided in admixtures with a modifying agentselected from the group consisting of cholesterols, stearyl amines,stearic acid, tocopherols, and mixtures thereof; and wherein theliposomes are unilamellar or multilamellar.
 13. The formulation of claim5, wherein the free anti-infective comprises between about 5% and about75% of the total of both the free and liposome-encapsulatedanti-infective; and wherein release of the total available amount ofencapsulated anti-infective occurs between about 2 hours and about 150hours.
 14. A method for treating a patient, comprising: aerosolizing aformulation comprising a free first pharmaceutically active drug and asecond pharmaceutically active drug encapsulated in liposomes; andinhaling the aerosol into the patient's lungs wherein the free drugcomprises between 10% and 70% a total of both free drug and encapsulateddrug in the formulation.
 15. The method of claim 14, wherein either theencapsulated pharmaceutically active drug is not an anti-infective andthe free pharmaceutically active drug is an anti-infective.
 16. Themethod of claim 15, wherein the free drug is ciprofloxacin and theencapsulated drug is a therapeutic nucleotide sequence.
 17. The methodof claim 14, wherein the liposomes have a diameter within a range offrom about 10 nm to about 10 μm and wherein said liposomes furthercomprise a lipid selected from the group consisting of fatty acids;lysolipids; sphingolipids; phosphatidylcholines;phosphatidylethanolamines; sphingomyelin; glycolipids; glucolipids;glycosphingolipids; phosphatidic acid; palmitic acid; stearic acid;arachidonic acid; oleic acid; lipids bearing sulfonated mono-, di-,oligo- or polysaccharides; lipids with ether and ester-linked fattyacids, polymerized lipids, diacetyl phosphate, stearylamine,cardiolipin, phospholipids with short chain fatty acids of 6 to 8carbons in length, synthetic phospholipids with asymmetric acyl chains;and lipids bearing a covalently bound polymer.
 18. The method of claim14, wherein the composition is comprised of the pharmaceutically activedrug in an amount of between 0.01% to 40% by weight.
 19. The method ofclaim 14, wherein the encapsulated pharmaceutically active drug isreleased at a rate of between about 0.1% to 100% per hour withsubstantially complete release of antibiotic occurring after about 1 to24 hours.
 20. The method of claim 14, wherein the encapsulatedpharmaceutically active drug is released at a rate of between about 0.5%to 20% per hour with substantially complete release of antibioticoccurring after about 1 to 12 hours.
 21. The method of claim 14, whereinthe encapsulated pharmaceutically active drug is released at a rate ofbetween about 2% to 10% per hour with substantially complete release ofantibiotic occurring after about 1 to 6 hours.
 22. The method of claim14, further comprising administering a therapeutic agent selected fromthe group consisting of DNase, a mucoregulatory agent, chemicals thatup-regulate the chloride ion channel or increase flow of ions across theepithelial surface of cells, including lantibiotics such as duramycin,agents to promote hydration or mucociliary clearance includingepithelial sodium channel (ENaC) inhibitors or P2Y2 agonists such asdenufosol, a bronchodilator, a steroid, an elastase inhibitor such asAlpha-1 antitrypsin (AAT), N-acetylcysteine, agents that enhance theactivity of an anti-infective against biofilm bacteria such as sodiumsalicylate, gamma interferon, alpha interferon, a fluoroquinolone, anantibiotic, or a fluoroquinolone selected from the group consisting ofamifloxacin, cinoxacin, ciprofloxacin, danofloxacin, difloxacin,enoxacin, enrofloxacin, fleroxacin, irloxacin, lomefloxacin, miloxacin,norfloxacin, ofloxacin, pefloxacin, rosoxacin, rufloxacin, sarafloxacin,sparfloxacin, temafloxacin and tosufloxacin or an antibiotic selectedfrom the group of tobramycin, colistin, azithromycin, amikacin, cefaclor(Ceclor), aztreonam, amoxicillin, ceftazidime, cephalexin (Keflex),gentamicin, vancomycin, imipenem, doripenem, piperacillin, minocycline,or erythromycin.
 23. A formulation, comprising: a pharmaceuticallyacceptable carrier for pulmonary delivery; a plurality of liposomes inthe carrier wherein the liposomes are comprised of a lipid andciprofloxacin; and a therapeutic nucleotide sequence.
 24. Theformulation of claim 23, wherein the nucleotide sequence in operativelyinserted in a vector and the liposomes are comprised of a compoundselected from the group consisting of poly-lactic acid, poly-glycolicacid, copolymers of lactic acid and glycolic acid and mixtures thereof;and a polymeric material selected from the group consisting ofcopolymers of lactic acid and glycolic acid, and mixtures thereof. 25.The formulation of claim 23, wherein the liposomes are comprised of acompound selected from the group consisting of methacralate,caprolactone, chitosan, calcium phosphate polyanhydrides, polyethyleneglycol, polyphosphoesters, polyetherester, polyphosphosphazenes,dextran, other carbohydrates or sugars, dendrimers or other starpolymers such as fullerenes, hyaluronic acid, collagen, gelatin, dextranor amphiphiles, lipids and various physical agglomerates of lipids withor without polymer hybrids.
 26. The formulation of claim 23, whereinsaid polymers, copolymers or conjugates are in a colloidal or suspensionform, a cross-linked form, present as crystals or nanocrystals, orpresent as calcium phosphate particles or nanoparticles.
 27. Theformulation of claim 23, wherein said formulation forms a depot.